I have adapted and optimized a novel approach to identify novel regulators during nuclear reprogramming. Induced pluripotent stem cells (iPSCs) hold great promise for modeling human diseases, drug discovery, and regenerative medicine. However, low iPSC reprogramming efficiency impede our ability to study the molecular mechanisms underlying these processes. Thus, heterokaryon, which are non-dividing, multinucleated fusion products of mouse embryonic stem cells and human fibroblasts, was developed here in the Blau laboratory to overcome these technical challenges. They are advantageous for studying mechanisms since: (i) reprogramming is fast and can be analyzed within minutes post-fusion; (ii) the efficiency of nuclear reprogramming is high (>70%); (iii) the onset of reprogramming is synchronous; and (iv) reprogramming can occur in the absence of cell division or DNA replication. I propose to use heterokaryons as a unique method to survey the dynamic transcriptional and epigenetic transitions necessary for down- regulation of the somatic program and acquisition of pluripotency. Specifically, I will address how AID and TET1 regulate active DNA demethylation to pluripotency. My proposed research addresses fundamental mechanisms underlying cellular plasticity and will create a paradigm for applying these principles toward directed differentiation and transdifferentiation.